Introduction to CellBiomaterial Engineering!
Module 3, Lecture 1!
20.109 Spring 2010!
Topics for Lecture 1!
•! Introduction to tissue engineering"
–! motivation"
–! basic principles + examples"
•! Introduction to Module 3"
–! background: cells and materials"
–! experiment: purpose and structure"
2!
Ability to repair tissue is limited!
•! Severe trauma (acute or disease-state)
challenges tissue repair capacity"
•! Donor tissue"
–! scarcity, immune response (graft or host)"
•! Autologous tissue"
–! availability, donor site morbidity"
•! Permanent synthetic substitute"
–! inammation, mis-match, failure "
•! A new approach: promote regeneration
of ~native tissue"
[Public domain image,
Wikimedia Commons]
3!
Tissue engineering: an emerging solution
!
“TE… applies the principles of
engineering and the life sciences !
toward the development of biological
substitutes that restore, maintain, or
improve tissue function.”!
-R. Langer & J.P. Vacanti, Science 260:920 (1993)!
What is in a tissue engineer!s toolkit?"
How good are the outcomes?!
Image by MIT OpenCourseWare. After Langer and Vacanti (1993).
[Langer & Vacanti]
4!
Scaffolds provide a framework!
•! Why a porous, degradable scaffold?"
–! mechanical support"
–! allow ingrowth, avoid inammation "
–! promote nutrient+oxygen diffusion"
•! How is the scaffold made degradable?"
–! cross-links susceptible to cleavage"
–! e.g., West JL & Hubbell JA, Macromolecules 32:341 (1999)"
collagenase exposure!
collagen peptide"
5!
Cytokines promote cell functions!
•! Types of cytokines"
–! growth factors (FGF, TGF, BMP)"
–! angiogenic (VEGF)"
–! chemokines (attract cells)"
•! Delivery of cytokines"
–! release from scaffold or transplanted cells"
•! Example: CCL21 promotes T cell migration "
Stachowiak et al., J Immunol 177:2340 (2006)."
Control
+CCL21!
!
6!
See supporting video, "Chemokinesis +CCL21."
See supporting video, "Chemokinesis control."
Courtesy of Darrell Irvine. Used with permission.
Cells make up tissues!
•! Progenitors vs. differentiated cells"
–! scarcity, function"
•! Transplanted vs. in situ cells"
–! scarcity, safety"
•! Example: tumor-inltrating
lymphocytes (TIL)"
tumor regression!
–! T cells lose function in tumors"
–! expand TIL ex vivo, treat with
cytokines, and transplant"
–! tested in mice!
Review: Rosenberg, et al. Nature Med 10:909 (2004). !
Data from: Overwijk, et al. J Exp Med 198:569 (2003).
7!
Source: Overwijk, W. W., et al. "Tumor Regression and Autoimmunity after Reversal of a
Functionally Tolerant State of Self-reactive CD8+ T Cells."
J Exp Med 198, no. 4 (August 18, 2003): 569. doi: 10.1084/jem.20030590
Courtesy of Willem Overwijk, et al., and Rockefeller University Press.
Components of a TE construct!
scaffold/matrix!
!" usually degradable, porous!
cells!
!" precursors and/or
differentiated!
!" often autologous!
soluble factors!
!" made by cells or synthetic!
!" various release proles!
integrated implantable
or injectable device!
8!
Putting it all together: in vitro construct!
See supporting video, "Cells in Scaffold."
Stachowiak et al. J Biomed Mater Res, 85A: 815 (2008)!
9!
Copyright © 2010 Wiley Periodicals, Inc., A Wiley Company. Reprinted with permission of John Wiley & Sons., Inc.
Supporting video for Stachowiak, A. N., and D. J. Irvine. "Inverse Opal Hydrogel-Collagen Composite Scaffolds as a Supportive Microenvironment
for Immune Cell Migration." J Biomed Mater Res 85A, no.3 (2008): 815-828.
Interlude: Shmeat!
http://www.colbertnation.com/the-colbertreport-videos/221975/march-17-2009/
world-of-nahlej---shmeat!
2:24 – 4:32!
Commercial success in TE!
•! Regenerating severely burned skin"
–! bilayer polymer [Yannas IV, et al. Science 215:174 (1982)]
•! top: protects wound, retains fluid
•! bottom: provides scaffold for growth
–! forms neotissue comparable to native skin
–! sold as Integra Dermal Regeneration template "
silicone
collagen-GAG
very thin
autograft
www.integra-ls.com/products/?product=46
Courtesy of Integra LifeSciences Corporation. Used with permission.
11!
Joint diseases: an unmet need!
•! Leading cause of physical disability in U.S. "
•! $100#s billion in in/direct costs"
Self-reported disease in
•! Osteoarthritis"
U.S., 2005
–! common in elderly population"
–! acute injury (athletes) !
susceptibility to early disease"
Muscle/bone!
Heart!
•! http://www.youtube.com/watch?
v=0dUSmaev5b0&feature=related"
Lung!
•! Limited pharma solutions"
–! pain management"
–! targets unknown"
–! cell therapies (Genzyme, Osiris)"
© 2008, American Academy of Orthopaedic Surgeons. Modified with permission from
The Burden of Musculoskeletal Diseases in the United States. Source of data: National
Center for Health Statistics, National Health Interview Survey, Adult Sample, 2005.
Our focus: cartilage tissue!
chondrocytes!
cartilage surface!
proteoglycans!
collagen bers!
boundary with bone!
Water-swollen, heterogeneous, avascular tissue.!
13!
Alginate: material for 3D culture!
•! Seaweed-derived polysacharride"
•! Co-polymer of M and G acids"
•! G-block polymer chains crosslinked by cations (e.g., Ca2+)"
•! Forms water-swollen gel"
calcium ions
G-block
M-block
•! G/M content and MW inuence"
–!
–!
–!
–!
mechanical properties"
swelling"
degradability"
viscosity of solution"
14!
Cells for cartilage TE!
Stem cells!
Chondrocytes!
Obtained from…!
Bone marrow!
Digested cartilage!
Recovery!
Difcult, initially very Easy, many cells!
few cells!
Expansion!
Many-fold!
Minimal!
Upkeep!
FGF to expand, TGF"1 to differentiate!
Multiple factors to
maintain phenotype!
stem cells!
chondrocytes!
broblasts!
15!
Specic goal and experiments!
•! Goal: examine effect of specic culture
conditions on chondrocyte phenotype"
Chondrocyte
culture!
•! Observe cell morphology and viability"
•! Measure collagen content"
–! Gene (RT-PCR) and protein (ELISA) expression"
–! Collagen II:I ratio reects cell state"
•! Grander purpose: cartilage TE"
–! conditions for ex vivo cell expansion"
–! conditions for in vitro cartilage production"
method 1!
method 2!
16!
Module overview: lab!
Day 1: design!
Day 4: prep RNA+cDNA!
Day 2: seed cultures!
Day 5: transcript assay!
Day 6: protein assay!
Day 7: remaining analysis!
Day 3: viability assay!
Day 8: your research ideas!!
17!
Lecture 1: conclusions!
•! Tissue engineering is an emerging interdisciplinary eld"
•! Maintaining cell function is a key part of TE"
•! Alginate beads provide a culture system for researching
soft tissues such as cartilage"
Next time… more about engineered and
natural biomaterials.!
18!
MIT OpenCourseWare
http://ocw.mit.edu
20.109 Laboratory Fundamentals in Biological Engineering
Spring 2010
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